phosphorus-radioisotopes and staurosporine-aglycone

We studied the phosphorylation of the secretory Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) in rat parotid acinar cells. We have previously shown that NKCC1 activity in these cells is dramatically upregulated in response to beta-adrenergic stimulation and that this upregulation correlates with NKCC1 phosphorylation, possibly due to protein kinase A (PKA). We show here that when ATP is added to purified acinar basolateral membranes (BLM), NKCC1 is phosphorylated as a result of membrane-associated protein kinase activity. Additional NKCC1 phosphorylation is seen when PKA is added to BLMs, but our data indicate that this is due to an effect of PKA on endogenous membrane kinase or phosphatase activities, rather than its direct phosphorylation of NKCC1. Also, phosphopeptide mapping demonstrates that these phosphorylations do not take place at the site associated with the upregulation of NKCC1 by beta-adrenergic stimulation. However, this upregulatory phosphorylation can be mimicked by the addition of cAMP to permeabilized acini, and this effect can be blocked by a specific PKA inhibitor. These latter results provide good evidence that PKA is indeed involved in the upregulatory phosphorylation of NKCC1 and suggest that an additional factor present in the acinar cell but absent from isolated membranes is required to bring about the phosphorylation.

Topics: Adenosine Triphosphate; Animals; Carbazoles; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Indole Alkaloids; Indoles; Isoproterenol; Isoquinolines; Maleimides; Marine Toxins; Microcystins; Okadaic Acid; Oxazoles; Parotid Gland; Peptides, Cyclic; Phenols; Phosphorus Radioisotopes; Phosphorylation; Rats; Sodium-Potassium-Chloride Symporters; Solute Carrier Family 12, Member 2; Staurosporine; Sulfonamides; Sympathomimetics; Vanadates

A 96-well microtiter enzyme-linked immunosorbent assay (ELISA) for protein tyrosine kinases has been developed. This assay uses one of several substrates that are phosphorylated by tyrosine kinase, an antibody to phosphotyrosine, and a peroxidase-linked second antibody. Color development is monitored by a change in absorbance at 450 nm and is dependent upon time, enzyme, ATP, and substrate concentrations. Specificity of the ELISA for phosphotyrosine was shown by inhibition of binding of the anti-phosphotyrosine antibody with phenyl phosphate. Results obtained in the ELISA compared favorably with those obtained by direct phosphorylation of the substrate with [32P]ATP. Staurosporine and K252a, protein kinase inhibitors, showed titratable inhibition of tyrosine kinase activity. This assay is a rapid, nonradioactive alternative to traditional methodology and is also amenable to automation.

Topics: Adenosine Triphosphate; Alkaloids; Antibodies; Autoanalysis; Binding, Competitive; Carbazoles; Enzyme Stability; Enzyme-Linked Immunosorbent Assay; ErbB Receptors; Indole Alkaloids; Muramidase; Phosphoric Monoester Hydrolases; Phosphorus Radioisotopes; Phosphorylation; Phosphotyrosine; Protein-Tyrosine Kinases; Staurosporine; Substrate Specificity; Tyrosine